Methods and assays for factor VIII activity

ABSTRACT

The methods and compositions described herein relate to the measurement of factor VIII (fVIII) levels and/or activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 15/036,199 filed May 12, 2016, which is a 35 U.S.C § 371 NationalPhase Entry Application of International Application No.PCT/US2014/064979 filed on Nov. 11, 2014, which designates the U.S. andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/904,948 filed Nov. 15, 2013, the contents of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to methods and assays for measuring factor VIIIactivity in a sample.

BACKGROUND

Factor VIII is a plasma glycoprotein that is essential for normalhemostasis. Defective or absent factor VIII causes hemophilia A, alife-threatening bleeding disorder. Because the gene for factor VIIIresides on the X chromosome, the disease occurs almost exclusively inmales, the sons of mothers with one defective factor VIII gene.

In the absence of therapy, hemophilia A is usually fatal prior to theage of reproduction. However, intravenous infusion of factor VIIIalleviates the bleeding risk. In developed countries patients areroutinely treated with pharmaceutical factor VIII so that the life-spanfor hemophilia A patients now approaches the life span of unaffectedpatients.

SUMMARY

The methods and assays described herein are based, in part, on thediscovery that platelets stimulated under physiological conditionscomprise binding sites for factor VIII (fVIII) that are independent ofphosphatidylserine. This differs from the stimulation of platelets by anon-physiological agonist, such as a calcium ionophore, which leads toexposure of many fVIII binding sites on the surface of platelets. Thus,provided herein are methods, assays, and kits for measuring fVIIIactivity under conditions that are more physiologically relevant. Inparticular, the replacement of phospholipid vesicles currently employedin the standard fVIII assays can provide more reliable measurement offVIII activity for clinical diagnosis of fVIII deficiency and monitoringof therapy with fVIII product infusions, as well as providing for moreaccurate assays of the activity of pharmaceutical preparations of fVIII,including engineered fVIII preparations, for which current assays areknown to have poor predictive value. The replacement of phospholipidvesicles having high phosphatidylserine concentration in the currentfVIII assays with platelet membrane-comprising or mimetic compositionsdescribed herein is also expected to enhance the reliability of fVIIIactivity remaining in individuals who express anti-fVIII antibodies.

In one aspect, described herein is a method of measuring fVIII activity,the method comprising contacting a sample in which fVIII is to bemeasured with fibrin or fibrinogen (fibrin(ogen)) and a plateletmembrane-comprising composition, under conditions that permit binding offibrin(ogen) to the platelet membrane-comprising composition and permitbinding of fVIII in the sample to the fibrin(ogen), and detectingactivity of fVIII.

In one embodiment, the platelet membrane-comprising compositioncomprises isolated platelets.

In another embodiment, the platelets are thrombin-activated platelets.

In another embodiment, the platelet membrane-comprising compositioncomprises a platelet membrane fraction comprising gpIIbIIIa (αII_(b)β3integrin).

In another embodiment, the platelet membrane-comprising compositioncomprises phospholipid vesicles.

In another embodiment, the contacting is performed in the presence ofFactor IXa (fIXa) and Factor X (fX).

In another embodiment, detecting activity of fVIII comprises detectingcleavage of a chromogenic fXa substrate by fXa.

In another embodiment, detecting activity of fVIII comprises aplasma-based clotting assay and/or use of a fibrometer and/or use of athromboelastometry device.

In another embodiment, detecting activity of fVIII bound to fibrin(ogen)is distinguished from fVIII that is not bound to fibrin(ogen) throughthe addition of a monoclonal antibody that selectively interferes withfVIII binding to fibrin(ogen).

In another aspect, described herein is a method of measuringpartitioning of fVIII between fibrin(ogen) and von Willebrand factor(vWf), the method comprising: contacting blood or plasma in whichpartitioning is to be measured with a solid support having vWf bindingmatrix immobilized thereupon under conditions that permit binding of vWfto fVIII; separately measuring fVIII activity bound to the solid supportvia vWF and fVIII activity remaining in suspension after the blood orplasma is contacted with the support, thereby determining the proportionof fVIII bound to vWF; contacting blood or plasma from the same sourcewith a solid support having fibrin(ogen) matrix immobilized thereuponunder conditions that permit binding of fibrin(ogen) to fVIII;separately measuring fVIII activity bound to the solid support viafibrin(ogen) and fVIII activity remaining in suspension after the bloodor plasma is contacted with the support, thereby determining theproportion of fVIII bound to fibrin(ogen); whereby the partitioning offVIII between vWf and fibrin(ogen) is measured.

In one embodiment, either or both of vWf and fibrin(ogen) areimmobilized to the solid support via an antibody that specifically bindsvWf or fVIII, respectively.

In another embodiment, the solid support comprises a column matrix.

In another embodiment, contacting with a solid support comprises passingthe sample over a column comprising the solid support.

In another embodiment, the solid support comprises agarose (e.g.,SEPHAROSE, GE Healthcare, Inc.) or polystyrene beads.

In another embodiment, after the contacting, the solid support is washedwith a buffer solution to remove unbound fVIII.

In another embodiment, washing of the solid support comprises a gradientsedimentation step.

In another aspect, described herein is a method of measuring thepartitioning of fVIII between binding to von Willebrand factor andbinding to fibrin(ogen) in a liquid sample, the method comprisingcontacting a solid support comprising fibrinogen or fibrin with thesample, removing the liquid sample from the solid support, and measuringfVIII activity bound to the support versus fVIII activity remaining inthe liquid sample, wherein fVIII bound to the support partitions withfibrin(ogen) and fVIII remaining in solution is free or partitions withvon Willebrand factor.

In one embodiment, either or both of vWf and fibrin(ogen) areimmobilized to the solid support via an antibody that specifically bindsvWf or fVIII, respectively.

In another embodiment, the solid support comprises a column matrix.

In another embodiment, contacting with the solid support comprisespassing the sample over a column comprising the solid support.

In another embodiment, the solid support comprises magnetic beads,agarose beads or polystyrene beads.

In another embodiment, after the contacting, the solid support is washedwith a buffer solution to remove unbound fVIII.

In another embodiment, washing of the solid support comprises a gradientsedimentation step.

In another aspect, described herein is a method of measuring thepartitioning of fVIII between binding to von Willebrand factor andbinding to fibrin(ogen) in a liquid sample, the method comprisingcontacting a solid support comprising an antibody that specificallybinds fVIII with the sample, removing the solid support, and measuringthe amount of von Willebrand factor and the amount of fibrin(ogen) boundto the support together with fVIII.

In one embodiment, the solid support comprises magnetic beads.

In another embodiment, the solid support comprises agarose beads.

In another embodiment, the solid support comprises polystyrene beads.

In another embodiment, the von Willebrand factor and fibrin(ogen) boundis measured by flow cytometry using fluorescently labeled antibodiesthat specifically bind von Willebrand factor and fibrin(ogen),respectively.

In another embodiment, the solid support is separated from the liquidsample by magnetic force.

In another embodiment, the solid support is separated from the liquid bysedimentation.

Also described herein are kits for measuring fVIII activity, the kitcomprising: fibrin(ogen); a platelet membrane-comprising composition;Factor IXa; Factor X; and packaging materials therefor.

In one embodiment, the kit further comprises a chromogenic fXasubstrate.

In another embodiment, the fibrin(ogen) is in association with theplatelet membrane-comprising composition.

In another embodiment, the platelet membrane-comprising compositioncomprises activated platelets.

In another embodiment, the platelet membrane-comprising compositioncomprises activated gpIIbIIIa incorporated into a membrane composition.

Also described herein are kits for detecting the fraction of fVIII boundto von Willebrand factor vs. fibrin(ogen) wherein the kits comprise: asolid support having monoclonal antibodies that specifically bind fVIIIimmobilized thereupon; a device for separating the beads from blood orplasma, fluorescently labeled detection antibodies that specificallybind fibrin(ogen) and von Willebrand factor, respectively; calibratedcontrol beads; and packaging materials therefor. The method ofseparating the beads from blood or plasma can comprise, e.g., a magnetor sedimentation.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B are line graphs showing binding of fluorescein-fVIII toplatelets in the presence of lactadherin. Platelets stimulated with 10μM A23187+ thrombin have many fVIII binding sites and approximately 95%are blocked by 200 nM lactadherin. Platelets stimulated through thethrombin pathway express 3% of binding sites relative to sites activatedby A23187. Lactadherin competes for approximately 25% of these siteswhile unlabeled fVIII competes for >70% of sites.

FIGS. 2A-2B are line graphs showing binding of fVIII 4-Ala to plateletsstimulated by 10 μM A23187+ thrombin or by thrombin alone. The number ofsites recognized by fVIII 4-Ala is reduced approximately 99% on A23187+thrombin-stimulated platelets but by approximately 40% onthrombin-stimulated platelets.

FIGS. 3A-3D are line graphs showing activity of fVIII 4-Ala on plateletsvs. phosphatidyl-L-serine (Ptd-L-Ser) vesicles. Platelets werestimulated with A23187 and thrombin (FIG. 3A) or thrombin alone (FIG.3C) and the supported activity of fVIII evaluated in a factor Xaseassay. By comparison, the activity of fVIII and fVIII 4-Ala wasevaluated in factor Xase assays supported by vesicles with 20% Ptd-L-Ser(FIG. 3B) or with 4% Ptd-L-Ser (FIG. 3D). fVIII 4-Ala supportsapproximately 5% residual activity on A23187+ thrombin-stimulatedplatelets, approximately 10% residual activity on thrombin-stimulatedplatelets. No residual activity was detected on vesicles with 20%Ptd-L-Ser or 4% Ptd-L-Ser.

FIGS. 4A-4C depict the binding of factor VIII to fibrin. FIG. 4A depictsa graph of experiments in which various concentrations of factorVIII-fluor were incubated with soluble fibrin immobilized onanti-fibrinogen-Superose beads. After 10 min. bound factor VIII wasevaluated by flow cytometry. Displayed results represent mean±range ofduplicates for a single experiment representative of 5 experiments.Saturable binding of factor VIII-fluor was observed. FIG. 4B depicts agraph of an experiment in which various concentrations of VWF wereincubated with 4 nM factor VIII-fluor for 15 min. prior to mixing withimmobilized fibrin-anti-fibrinogen-Superose beads. Data are from asingle experiment representative of 5 experiments. VWF inhibited factorVIII binding to fibrin. FIG. 4C depicts a graph of an experiment inwhich various concentrations of factor VIII-C2 were incubated withfactor VIII-fluor prior to mixing with fibrin-anti-fibrinogen-Superosebeads. Experiments were performed in tris-buffered saline containing0.02 M NaCl. Factor VIII-C2 competed with factor VIII-fluor for bindingto immobilized fibrin. Results are from a single experimentrepresentative of three experiments. Displayed data are corrected formeasured background fluorescence with control beads lacking fibrin

FIGS. 5A-5B show the effect of fibrin on function of fVIII. (FIG. 5A)Various concentrations of fibrinogen were mixed with fVIII, factor IXa,factor X, and phospholipid vesicles prior to the simultaneous additionof thrombin (1 u/ml) and Ca⁺⁺ (1.5 mM). The reaction was stopped afterthe minutes by addition of EDTA and factor Xa measured with chromogenicsubstrate S-2765. Fibrin increased activity approx. 3.5-fold. The degreeof enhancement of factor Xase activity by 20 μg/ml fibrin was evaluatedwith phospholipid vesicles of varying phosphatidylserine (PS) content(FIG. 5B). All vesicles had 10% PE with the balance as PC. Activity isdisplayed as the ratio of activity with fibrin/activity without fibrinEnhancement was greatest with 4% PS vesicles.

FIGS. 6A-6B demonstrate the effect of anti-factor VIII mAb's on factorVIII binding to fibrin. FIG. 6A depicts a graph of an experiment inwhich factor VIII-fluor, 4 nM, was incubated with 0.75 μg/ml ESH4 or0.75 μg/ml ESH8 for 1 hr prior to mixing withfibrin-anti-fibrin-Superose or control beads lacking fibrin. Results arefrom a single experiment representative of 3 experiments. ESH4 and ESH8decreased binding to below control levels (*) observed when factorVIII-fluor was incubated with control Superose beads. FIG. 6B depicts agraph of an experiment in which factor VIII was incubated with 10 μg/mLESH4 or ESH8 for 1 hr at 23° C. prior to addition of factor IXa, factorX, thrombin, and Ca⁺⁺ as indicated in the legend to FIGS. 5A-5B. In theabsence of antibodies, addition of 10 μg/ml fibrin increased Xaseactivity about 2-fold. Fibrin did not increase activity in the presenceof ESH4 or ESH8 above the levels observed in the absence of fibrin.Results are from a single experiment representative of 4 experiments(FIG. 6A) and are mean±SD for 4 experiments. FIG. 6B is a bar graphshowing the effect of anti-fVIII monoclonal antibodies on fVIII bindingto fibrin and fibrin enhancement of factor Xase function.

FIGS. 7A-7C demonstrate the effect of ESH4 and ESH on factor VIIIbinding to thrombin-stimulated platelets. FIG. 7A depicts a graph of anexperiment in which factor VIII-fluor was incubated with 14 μg/ml ESH8prior to mixing with platelets stimulated by thrombin. Bound factor VIIIwas measured by flow cytometry. ESH8 decreased bound factor VIII byapprox. 70%. FIG. 7B depicts a graph of an experiment in which variousconcentrations of ESH4 were incubated with 2 nM factor VIII-fluor for 5min prior to addition of thrombin-stimulated platelets. Bound factorVIII was evaluated by flow cytometry after 5 min. ESH4 inhibited approx.80% of factor VIII binding with a half-maximal effect at <0.5 μg/ml.FIG. 7C depicts a graph of an experiment in which ESH8 or ESH4, 10 μg/mlwas mixed with factor VIII for 60 min. prior to addition of factor IXa,factor X, platelets, Ca++, and thrombin at the concentrations indicatedabove. ESH8 inhibited 84% of activity and ESH4 inhibited 78% ofactivity. To obtain average aggregate values, factor Xase activity wasnormalized to the value in the absence of ESH4 or ESH8 for eachexperiment. Results are from a single experiment representative of twoexperiments (FIG. 7A) and 3 experiments (FIG. 7B). FIG. 7C is mean±SDfrom 4 experiments each performed in duplicate.

FIG. 8 is a graph showing the amount of bound fVIII at differentconcentrations of fibrinogen. FVIII was incubated withthrombin-stimulated platelets in the presence and absence of variousconcentrations of fibrin(ogen). Increased concentrations of fibrinogendecreased fVIII bound to platelets.

FIG. 9 is a graph showing the amount of factor XA formed at increasingconcentrations of fibrin. Platelets were stimulated with thrombin andthe thrombin blocked by hirudin. The platelet-supported factor Xaseactivity was measured in the presence of fVIII, factor IXa and factor X.The reaction was initiated by the addition of 1 nM factor Xa andterminated after 10 min by addition of EDTA. Factor Xa formed wasmeasured with chromogenic substrate. Lower concentrations of fibrinincreased factor Xase activity while higher concentrations decreasedactivity.

FIGS. 10A-10C demonstrate the effect of ESH4 and ESH8 on activity offactor VIII with activated platelets and phospholipid vesicles. FIG. 10Adepicts a graph of an experiment in which inhibition of 1 unit/ml factorVIII activity by ESH4 and ESH8 was evaluated against a factor VIIIconcentration curve in re-constituted platelet-rich-plasma. Fibrinformation was initiated by addition of 70 pM factor XIa, thrombinactivation peptides, and Ca++. A log linear plot demonstratessensitivity to factor VIII concentration over a 4-log range in factorVIII concentration. ESH4 inhibited somewhat more activity than ESH8.Results are representative of 3 experiments performed in full and 5performed with fewer factor VIII concentrations. FIG. 10B depicts agraph comparing residual factor VIII activity in various plasma-basedactivities. aPTT and 2-stage results represents mean±SD for publishedresults in reference (44) weighted equally with results obtained in thislaboratory using commercial aPTT and 2-stage reagents. Inhibition wasalso evaluated in reconstituted platelet-rich-plasma lacking vonWillebrand factor (act Platelet (−VWF)). Results from this lab are from4 experiments (aPTT, 2-stage), activated platelets mean±SD for 3experiments, activated platelets w/o von Willebrand factor −1experiment. The inset, with log x-scale illustrates the error bars andallows comparison with and without von Willebrand factor. FIG. 10Cdepicts a bar graph comparing residual factor VIII activity in thepresence of 10 μg/ml ESH8. aPTT without von Willebrand factor (aPTT(−VWF)) is from reference (32). Values on activated platelets aremean±SEM for 2 experiments and 1 experiment for plasma lacking vonWillebrand factor.

DETAILED DESCRIPTION

In one aspect, provided herein are methods, assays and kits formeasuring fVIII activity in a sample. The methods and assays rely on thephysiological activation of platelets by e.g., thrombin for exposingsites on the surface of platelets that bind fVIII independently ofphosphatidylserine (e.g., platelet-bound fibrin or fibrinogen). Theinventors have discovered, in part, that platelet stimulation by acalcium ionophore (a non-physiological agonist) leads to exposure ofmany binding sites comprised primarily of exposed phosphatidylserine,whereas platelets stimulated by thrombin (a physiological agonist) havebinding sites that are comprised of platelet-bound fibrin or fibrinogen.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

“Factor VIII activity” or “fVIII activity” is defined as the ability tofunction in the coagulation cascade, induce the formation of Factor Xavia interaction with Factor IXa on an activated platelet, and supportthe formation of a blood clot.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Hemophilia A

Hemophilia A is an X-linked hemorrhagic disorder resulting frommutations in the gene encoding fVIII. Affected individuals commonlypresent spontaneous hemorrhages and prolonged bleeding after trauma orsurgery. Severely affected subjects present with levels of fVIII thatare lower than 1% of normal and comprise the majority of clinicallysymptomatic cases. The remaining patients have mild to moderate diseasewith factor levels of 1-30%. In this application, “Hemophilia A” refersto patients with less than 1% fVIII unless otherwise specified.

People with deficiencies in fVIII who are not treated with fVIII cansuffer uncontrolled internal bleeding that may cause a range of serioussymptoms, from inflammatory reactions in joints to early death. Severehemophiliacs can be treated with infusion of human fVIII, which willrestore the blood's normal clotting ability if administered withsufficient frequency and concentration. The classic definition of fVIII,in fact, is that substance present in normal blood plasma that correctsthe clotting defect in plasma derived from individuals with hemophiliaA.

Factor VIII (fVIII)

FVIII functions in an enzyme complex that converts the pro-enzyme,factor X, to the enzyme, factor Xa. FVIII, a pro-cofactor, is cleaved atat least two peptide bonds, to become an active cofactor (fVIIIa).FVIIIa binds to sites on a cell membrane and forms a complex with factorIXa, a serine proteinase. The membrane-bound complex of fVIIIa withfactor IXa cleaves factor X with an efficiency that is at least10,000-fold greater than factor IXa alone. The current literatureindicates that phosphatidylserine is a critical constituent of fVIIIabinding sites.

FVIII circulates in plasma in complex with von Willebrand factor (vWf).Binding to von Willebrand factor decreases the rate at which fVIII iscleared from plasma and decreases the susceptibility of fVIII todegradation by enzymes within plasma. FVIII can dissociate from vonWillebrand factor in three ways, prior to gaining activity. First, fVIIIdissociates from von Willebrand factor in a slow dissociation process tomaintain an equilibrium dissociation constant of 0.1-0.2 nM. Second,fVIII dissociates more rapidly from von Willebrand factor that has boundto collagen or other molecules. Third, fVIII is cleaved by thrombin at ascissile bond at amino acid 1689 of fVIII. Upon cleavage oat this site,an acidic peptide is released and fVIII dissociates rapidly from vonWillebrand factor. Provided herein are methods and assays based on thediscovery that a second plasma protein, fibrinogen, binds fVIII and thatfVIII in complex with fibrinogen cannot bind von Willebrand factor.

Factor VIII Assays

Current standard practice for measuring fVIII activity typically uses a“one-stage” or a “two-stage” assay.

One stage assays rely on mixture of subject plasma with plasma that isdeficient in fVIII. The plasma is incubated with an activator and thencalcium is added to initiate coagulation. The length of time until clotformation is inversely related to the quantity of fVIII. The assay canbe used to evaluate the factor activity of pharmaceutical fVIIIconcentrations. The fVIII product is diluted to various degrees andmixed with fVIII deficient plasma. In either case, the quantity of fVIIIis evaluated by comparison to dilutions of normal plasma (with fVIII).

Two stage assays rely on mixture of subject plasma with a mixturecontaining factor IXa (fIXa), factor X (fX) and phospholipid vesicles.Calcium is added to permit the fVIIIa-fIXa complex to form and activatefX to fXa. After an interval, the reaction is quenched. The quantity offX formed, proportional to fVIII activity, is measured by the rate atwhich a chromogenic substrate is cleaved by fXa.

Both assay types rely on phospholipid vesicles to provide a membranesurface on which the fVIIIa-fIXa complex assembles. The composition ofthe phospholipid mixture is proprietary for some preparations, but isgenerally know to be comprised of a mixture of phosphatidylserine,phosphatidylcholine, and phosphatidylethanolamine. The one and two-stageassays described above provide different values for activity ofengineered products. This has led to uncertainty in the biologicactivity and the appropriate doses for patients. This problem has beenrecognized by the FDA, the NIH, and pharmaceutical companies.

Such assays are described in e.g., Manucci and Tripodi, “Factor VIIIclotting activity”. E.C.A.T. assay procedures, London: Kluwer AcademicPublishers, 1999; endogenous thrombin potential analysis, as describedin Hemker et al., “The thrombogram: monitoring thrombin generation inplatelet-rich plasma,” Thrombosis and haemostasis, vol. 83:589-591,among others.

Fibrinogen

Fibrinogen is a major plasma protein, present at approximately 200-foldhigher concentration than von Willebrand factor. Fibrinogen has severalproven physiologic roles. Thrombin cleaves fibrinogen to form fibrin andthe fibrin molecules self assemble to form a gel, the materialresponsible for physical properties of a blood clot. Fibrinogen is acritical ligand for the α_(IIb)β₃ integrin of platelets, functioning asa bridge between platelets as they aggregate. Fibrin has known bindingaffinity for thrombin, vitronectin, and plasmin and these proteinsinfluence the durability of blood clots. Fibrinogen is commerciallyavailable, e.g., from Sigma-Aldrich (see, e.g., Catalog Nos. F38799 andF4883, both human fibrinogen products).

Other Clotting Factors, Substrates and Equipment

Other factors of use in the methods, compositions and kits describedherein include, for example, human fIX and fIXa. Human fIX iscommercially available, e.g., from Alpha Therapeutic (ALPHANINE SD™),Pfizer (BENEFIX™), CSL Behring (MONONINE™). fIX is converted to fIXa inplasma.

Human fX is commercially available, e.g., from Sino Biologicals, Inc.,Daxing, China (see, e.g., catalog No. 11076-H08B and No. HG-11076-M) andfrom Enzyme Research Laboratories, Inc. (see, e.g., catalog No. HFX1010).

Chromogenic fXa substrates useful in assays as described herein arecommercially available. For example, Sigma-Aldrich sells chromogenic fXasubstrate CH₃OCO-D-CHA-Gly-Arg-pNA-AcOH under Catalog No. F-3301, and athrombin generation chromogenic substrate, β-Ala-Gly-Arg-p-nitroanilidediacetate under Catalog No. T3068. Chromogenix (Milan, IT) sells achromogenic fXa substrate as Catalog No. S-2765. Abcam (Cambridge,Mass.) also sells a Factor X Human Chromogenic Activity Assay kit underCatalog No. ab108833, which measures activated fX in plasma and culturemedium, among other sample types.

Monoclonal antibodies specific for fibrin(ogen) and for vWf arecommercially available, e.g., from Santa Cruz Biotech. Kits for couplingantibodies and other peptide or polypeptide agents to solid supports,e.g., beads, are also commercially available, e.g., from LifeTechnologies, Inc. and from Abcam, Inc.

Block Scientific sells the BBL Fibrometer coagulation analyzer made byBD Diagnostic Systems.

Thromboelastometry provides another method of measuring clotting and theeffects of clotting factors. Thromboelastometry, also referred to asrotational thromboelastometry or rotational thromboelastography, is aviscoelastic method for haemostasis testing in whole blood, and permitsinvestigation of the interaction of coagulation factors, theirinhibitors, anticoagulant drugs, blood cells, specifically platelets,during clotting and subsequent fibrinolysis. The rheological conditionsmimic the sluggish flow of blood in veins. Thromboelastometry isperformed, for example, with the ROTEM whole blood analyzer (TemInnovations GmbH, Munich). For such assays, blood (300 μl,anticoagulated with citrate) is placed into a cuvette. A disposable pinis attached to a shaft which is connected with a thin spring and slowlyoscillates back and forth. The signal of the pin suspended in the bloodsample is transmitted via an optical detector system. The test isstarted by adding appropriate reagents. The instrument measures andgraphically displays the changes in elasticity at all stages of thedeveloping and resolving clot.

Platelet Membrane-Comprising Compositions:

Various methods and compositions described herein use plateletmembrane-comprising compositions that permit the assembly of an activeenzyme complex that activates fX to fXa. The membrane-comprisingcompositions applicable to the methods and compositions described hereinhave the ability to bind fibrin or fibrinogen, e.g., via the gpIIbIIIaglycoprotein complex associated with the membrane composition, which isa platelet receptor for fibrinogen. The activated complex is formed bythe association of glycoprotein IIb with glycoprotein Ma in the contextof a phospholipid membrane. As such, platelet membrane-comprisingcompositions as described herein can include whole platelets, e.g.,human donor platelets, including thrombin-activated platelets, as wellas membrane fractions made from them that include the gpIIbIIIa complexand retain the ability to bind fibrinogen. Methods for the isolation ofplatelets and platelet membrane fractions are well known. As oneexample, Heijnen et al., Blood 94: 3791-3799 (1999), which isincorporated herein by reference in its entirety, describes methods ofplatelet isolation. This reference also describes membrane microvesiclesreleased from activated platelets that are enriched for glycoproteinsand support the formation of the fX/prothrombin complex. The referencedescribes how to isolate the membrane microvesicles, as well as analysisof them using flow cytometry. Microvesicles of the kind described byHeijnen et al. are specifically contemplated for use as plateletmembrane-comprising compositions as needed in methods and compositionsas described herein. Other platelet membrane-containing fractions thatsupport the methods and compositions described herein include, forexample, the platelet-derived microparticles described by Gilbert etal., J. Biol. Chem., 266: 17261-17268 (1991), also incorporated hereinby reference in its entirety.

It is contemplated that rather than requiring the phospholipids beisolated from platelets, synthetic or purified phospholipids can beformulated in association with gpIIbIIIa and also be used in methods andcompositions as described herein in place of membrane compositionsisolated from platelets. The critical factor for such compositions,which mimic platelet membranes (“platelet membrane mimetics”) is thatthey permit binding of fibrin to gpIIbIIIa, and that the phospholipidused contains at least 2% phosphatidylserine. Among other possibilities,the gpIIbIIIa/phospholipid compositions can be formulated, e.g., asliposomes of nanodiscs. The preparation of liposomes and nanodiscscomprising active gpIIbIIIa is described, for example, by Ye et al. J.Cell Biol. 188: 157-173 (2010), which is incorporated herein byreference in its entirety. It is also contemplated, for example, thatother membrane-associated proteins, engineered to specifically bindfibrinogen, e.g., by inclusion of an antibody domain that bindsfibrinogen, could also support the assembly of the fVIII-fibrinogencomplex involved in the fVIII assay methods described herein.

Blood Coagulation Assays

Blood coagulation (clotting) assists homeostasis by minimizing bloodloss. In vivo, clotting usually requires vessel damage, plateletaggregation, coagulation factors and inhibition of fibrinolysis. Thecoagulation factors have been reported to act through a cascade thatrelates vessel damage to formation of a blood clot. See generally L.Stryer, Biochemistry, 3rd Ed, W. H. Freeman Co., New York; A. G. Gilmanet al., The Pharmacological Basis of Therapeutics, 9th Edition, McGrawHill Inc., New York, pp. 1341-1359; and Mann, K. G. et al. (1992) Semin.Hematol. 29:213.

Initiation of blood coagulation arises from two distinct pathways: theintrinsic (contact) and extrinsic pathways. The intrinsic pathway can betriggered in vitro by contact of blood borne factor with artificialnegatively charged surfaces such as glass. In contrast, the extrinsicpathway can be initiated in vivo or in vitro when tissue factor (TF) ona phospholipid surface, normally sequestered from the circulatorysystem, comes into contact with blood following injury. Both pathwaysare characterized by the assembly of multiple protein complexes onprocoagulant surfaces, which serves to localize the response to the siteof injury. See e.g, Mann, K. G. et al. (1990) Blood 76: 1; Mann, K. G.et al. (1992), supra.

Current theories of coagulation maintain that an interplay between thetwo pathways is required for efficient blood clotting. See e.g., S. I.,Rapaport and L. V. M. Rao (1995) Throm. Haemost. 74: 7. The contactpathway has been further divided into early and late steps. These stepsare typically associated with specific coagulation factors. It has beenreported that hemophilia A, B and C are each correlated withdeficiencies in the late contact pathway (fVIII, fIX, and fXI,respectively).

Many activities of the extrinsic and intrinsic tenases (fVIIIa-fIXa) andthe prothrombinase complex are facilitated by activated platelets andother phospholipid membranes.

In clinical settings, citrated plasma isolates are the most widely usedblood product for coagulation testing, due to prominence of theprothrombin time (PT) and activated partial thromboplastin time (aPTT)tests. The PT is the more convenient assay, and is performed by additionof a large quantity of thromboplastin to the citrated plasma, withsubsequent initiation of the reaction by calcium addition. The time toclot formation is noted, which for most normal donors is typically about10 to about 14 seconds. The aPTT test involves about a 3 to about 5minute preincubation of the citrated plasma with a mixture ofphospholipids and solids possessing negatively charged surfaces. Thereaction is initiated by calcium addition, and the clot time for normaldonors typically falls between 25 and 43 seconds. While well establishedin the clinical venue, neither assay is entirely suitable to mimic thephysiological coagulation reaction in its entirety.

For example, while the PT measurement employs the physiologicallyrelevant initiator TF and the assay is sensitive to Factors V, VII, X,and prothrombin (II), the concentration used is sufficiently high thatthe reaction is usually insensitive to deficiencies or abnormalities incoagulation Factors VIII or IX. Clotting occurs rapidly in normalindividuals (about 10 to about 14 seconds), and errors in measurement onthe order of seconds are a significant fraction of the total clot time.The PT is an effective assay for measuring clotting time of a bloodsample.

The aPTT assay is also associated with problems. For example, sinceinitiation proceeds through the early contact pathway members, FactorVII is bypassed in this reaction. As a result, this assay in insensitiveto deficiencies or abnormalities in this biologically importantcoagulation factor. Additionally, most aPTT assays use plasma and arenot compatible with whole blood. When the assay is used to monitoradministration of anti-coagulants, the target range for prolongation ofthe clot time is between about 2.5 to about 3.5 times normal, or betweenabout 25 and 49 seconds. There has been recognition that this time rangeis often too small to permit accurate analysis.

Solid Supports

In certain embodiments, it is desirable to measure the partitioning offVIII between fibrinogen and von Willebrand factor. In one embodiment,von Willebrand factor is immobilized to a solid support. In anotherembodiment, fibrin or fibrinogen is immobilized to a solid support. Inother embodiments, antibodies that specifically bind one factor/proteinor another are immobilized on a solid support. Protein or peptideimmobilization can be achieved using methods routine to those ofordinary skill in the art, and can be direct, e.g., by binding of theprotein or peptide directly to the surface of the support, or, indirect,e.g., by binding through a linker (peptide or other chemical-basedlinkers) or by binding to an antibody immobilized on the support surfacethat recognizes and binds the desired factor or protein. In someembodiments, indirect binding provides advantages in activity of thebound protein/factor—while not wishing to be bound by theory, sucheffects are likely due to improved access of the linked factor orprotein to its environment and accompanying lack of steric hindrances.

In some embodiments, the solid support comprises, for example, magneticbeads, SEPHAROSE or other agarose beads, a nitrocellulose membrane, anylon membrane, a column chromatography matrix, a high performanceliquid chromatography (HPLC) matrix or a fast performance liquidchromatography (FPLC) matrix for purification. As used herein, the term“magnetic bead” means any solid support that is attracted by a magneticfield; such solid supports include, without limitation, DYNABEADS™ (LifeTechnologies, Inc.), BIOMAG™ (Qiagen, Inc.), MPG7 (PureBiotech LLC),MAGNESPHERE™ (Promega, Inc.) Magnetic Particles, any of the MAGNA™ lineof magnetizable particles (Millipore, Inc.), BIOMAG™ SuperparamagneticParticles (Qiagen, Inc.), or any other magnetic bead to which a molecule(e.g., a protein factor, fibrin(ogen) or antibody) may be attached orimmobilized. In one embodiment, a magnet or magnetic field can be usedto separate the target protein or factor, and those other proteins orfactors complexed with it, from bulk solution or suspension.

Screening Assays

Screening assays as contemplated herein can be used to identifymodulators, i.e., candidate or test compounds or agents (e.g., peptides,antibodies, peptidomimetics, small molecules (organic or inorganic) orother drugs) which modulate fVIII activity. In certain aspects a screenfor agents that modulate the interaction between fVIII and fibrin(ogen)are of interest.

The term “candidate agent” is used herein to mean any agent that isbeing examined for ability to modulate the activity of fVIII. Althoughthe method generally is used as a screening assay to identify previouslyunknown molecules that can act as a therapeutic agent, or an agent thatmodifies fVIII activity or fVIII interactions with fibrin(ogen), thescreening described herein can also be used to confirm that an agentknown to have such activity, in fact has the activity, for example, instandardizing the activity of the therapeutic agent. A candidate agentcan be any type of molecule, including, for example, a peptide, apeptidomimetic, a polynucleotide, or a small organic molecule, that onewishes to examine for the ability to modulate a desired activity, suchas, for example, increasing or prolonging fVIII activity. It will berecognized that the screening methods described herein are readilyadaptable to a high throughput format and, therefore, the methods areconvenient for screening a plurality of test agents either serially orin parallel. The plurality of test agents can be, for example, a libraryof test agents produced by a combinatorial method library of testagents. Methods for preparing a combinatorial library of molecules thatcan be tested for fVIII-modulating activity are well known in the artand include, for example, methods of making a phage display library ofpeptides, which can be constrained peptides (see, for example, U.S. Pat.Nos. 5,622,699; 5,206,347; Scott and Smith, Science 249:386-390, 1992;Markland et al., Gene 109:1319, 1991; each of which is incorporatedherein by reference in their entireties); a peptide library (U.S. Pat.No. 5,264,563, which is incorporated herein by reference); apeptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:8392,1995; a nucleic acid library (O'Connell et al., supra, 1996; Tuerk andGold, supra, 1990; Gold et al., supra, 1995; each of which isincorporated herein by reference in their entireties); anoligosaccharide library (York et al., Carb. Res., 285:99128, 1996; Lianget al., Science, 274:1520-1522, 1996; Ding et al., Adv. Expt. Med.Biol., 376:261-269, 1995; each of which is incorporated herein byreference in their entireties); a lipoprotein library (de Kruif et al.,FEBS Lett., 399:232-236, 1996, which is incorporated herein by referencein their entireties); a glycoprotein or glycolipid library (Karaoglu etal., J. Cell Biol., 130:567-577, 1995, which is incorporated herein byreference); or a chemical library containing, for example, drugs orother pharmaceutical agents (Gordon et al., J. Med. Chem., 37:1385-1401,1994; Ecker and Crooke, Bio/Technology, 13:351-360, 1995; each of whichis incorporated herein by reference in their entireties).

Accordingly, the term “agent” as used herein in the context of screeningmeans any compound or substance such as, but not limited to, a smallmolecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An “agent”can be any chemical, entity or moiety, including without limitationsynthetic and naturally-occurring proteinaceous and non-proteinaceousentities. In some embodiments, an agent is nucleic acid, nucleic acidanalogues, proteins, antibodies, peptides, aptamers, oligomer of nucleicacids, amino acids, or carbohydrates including without limitationproteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs,lipoproteins, aptamers, and modifications and combinations thereof etc.In some embodiments, the nucleic acid is DNA or RNA, and nucleic acidanalogues, for example can be PNA, pcPNA and LNA. A nucleic acid may besingle or double stranded, and can be selected from a group comprising;nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc.Such nucleic acid sequences include, for example, but are not limitedto, nucleic acid sequence encoding proteins that act as transcriptionalrepressors, antisense molecules, ribozymes, small inhibitory nucleicacid sequences, for example but not limited to RNAi, shRNAi, siRNA,micro RNAi (mRNAi), antisense oligonucleotides etc. A protein and/orpeptide agent or fragment thereof, can be any protein of interest thatmodulates fVIII activity or pharmacokinetics, for example, but notlimited to; mutated proteins; therapeutic proteins; truncated proteins,wherein the protein is normally absent or expressed at lower levels inthe cell. Proteins of interest can be selected from a group comprising;mutated proteins, genetically engineered proteins, peptides, syntheticpeptides, recombinant proteins, chimeric proteins, antibodies, humanizedproteins, humanized antibodies, chimeric antibodies, modified proteinsand fragments thereof.

In certain embodiments, the candidate agent is a small molecule having achemical moiety. Such chemical moieties can include, for example,unsubstituted or substituted alkyl, aromatic, or heterocyclyl moietiesand typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups,including macrolides, leptomycins and related natural products oranalogues thereof. Candidate agents can be known to have a desiredactivity and/or property, or can be selected from a library of diversecompounds. Also included as candidate agents are pharmacologicallyactive drugs, genetically active molecules, etc. Such candidate agentsof interest include, for example, chemotherapeutic agents, hormones orhormone antagonists, growth factors or recombinant growth factors andfragments and variants thereof. Exemplary of pharmaceutical agentssuitable for use with the screening methods described herein are thosedescribed in, “The Pharmacological Basis of Therapeutics,” Goodman andGilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under thesections: Water, Salts and Ions; Drugs Affecting Renal Function andElectrolyte Metabolism; Drugs Affecting Gastrointestinal Function;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists;Vitamins, Dermatology; and Toxicology, all of which are incorporatedherein by reference in their entireties. Also included are toxins, andbiological and chemical warfare agents, for example see Somani, S. M.(Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992), thecontents of which is herein incorporated in its entirety by reference.Candidate agents, such as chemical compounds, can be obtained from awide variety of sources including libraries of synthetic or naturalcompounds, such as small molecule compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds, including biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and canbe used to produce combinatorial libraries. Known pharmacological agentscan be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Synthetic chemistry transformations and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe candidate compounds for use in the screening methods describedherein are known in the art and include, for example, those such asdescribed in R. Larock (1989) Comprehensive Organic Transformations, VCHPublishers; T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995), and subsequent editions thereof,the contents of each of which are herein incorporated in theirentireties by reference. Examples of methods for the synthesis ofmolecular libraries can be found in the art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994)Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med.Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem.Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233,the contents of each of which are herein incorporated in theirentireties by reference. Libraries of candidate agents can also, in someembodiments, be presented in solution (e.g., Houghten (1992),Biotechniques 13:412-421), or on beads (Lam (1991), Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull etal. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406;Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991)J. Mol. Biol. 222:301-310; Ladner supra.), the contents of each of whichare herein incorporated in their entireties by reference. The testcompounds or candidate agents can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145). Examples of methods for the synthesis of molecular librariescan be found in the art, for example in: DeWitt et al. (1993) Proc.Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad.Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Choet al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int.Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries ofcompounds can be presented in solution (e.g., Houghten (1992)Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al.(1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith(1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.). The methodsdescribed herein further pertain to novel agents identified by theabove-described screening assays.

Screening assays can be performed, for example, by performing a fVIIIactivity assay as described herein with a known or constant amount ofactive fVIII in the presence and absence of a candidate agent. Adifference in fVIII activity in such an assay is indicative that thecandidate agent modifies the process assayed. Varying the amount or type(e.g., wild-type, mutated, engineered) of fVIII used can determinewhether the effect is directly upon fVIII or indirect.

It is understood that the foregoing description and the followingexamples are illustrative only and are not to be taken as limitationsupon the scope of the invention. Various changes and modifications tothe disclosed embodiments, which will be apparent to those of skill inthe art, may be made without departing from the spirit and scope of thepresent invention. Further, all patents, patent applications, andpublications identified are expressly incorporated in their entiretiesherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method of measuring Factor VIII (fVIII) activity, the        method comprising contacting a sample in which fVIII is to be        measured with fibrin or fibrinogen (fibrin(ogen)) and a platelet        membrane-comprising composition, under conditions that permit        binding of fibrin(ogen) to the platelet membrane-comprising        composition and permit binding of fVIII in the sample to the        fibrin(ogen), and detecting activity of the fVIII.    -   2. The method of paragraph 1 wherein the platelet        membrane-comprising composition comprises isolated platelets.    -   3. The method of paragraph 2 wherein the platelets are        thrombin-activated platelets.    -   4. The method of paragraph 1 wherein the platelet        membrane-comprising composition comprises a platelet membrane        fraction comprising gpIIbIIIa.    -   5. The method of paragraph 1 wherein the platelet        membrane-comprising composition comprises phospholipid vesicles.    -   6. The method of paragraph 1 wherein the contacting is performed        in the presence of Factor IXa (fIXa) and Factor X (fX).    -   7. The method of paragraph 1 wherein detecting activity of fVIII        comprises detecting cleavage of a chromogenic fXa substrate by        fXa.    -   8. The method of paragraph 1 wherein detecting activity of fVIII        comprises a plasma-based clotting assay and/or use of a        fibrometer and/or use of a thromboelastometry device.    -   9. The method of paragraph 1 wherein detecting activity of fVIII        bound to fibrin(ogen) is distinguished from fVIII that is not        bound to fibrin(ogen) through the addition of a monoclonal        antibody that selectively interferes with fVIII binding to        fibrin(ogen).    -   10. A method of measuring partitioning of fVIII between        fibrinogen and von Willebrand factor (vWf), the method        comprising:    -   contacting blood or plasma in which partitioning is to be        measured with a solid support having vWf immobilized thereupon        under conditions that permit binding of vWf to fVIII;    -   separately measuring fVIII activity bound to the solid support        via vWF and fVIII activity remaining in suspension after the        blood or plasma is contacted with the support, thereby        determining the proportion of fVIII bound to vWF;    -   contacting blood or plasma from the same source with a solid        support having fibrin(ogen) immobilized thereupon under        conditions that permit binding of fibrin(ogen) to fVIII;    -   separately measuring fVIII activity bound to the solid support        via fibrin(ogen) and fVIII activity remaining in suspension        after the blood or plasma is contacted with the support, thereby        determining the proportion of fVIII bound to fibrin(ogen);    -   whereby the partitioning of fVIII between vWf and fibrin(ogen)        is measured.    -   11. The method of paragraph 10 wherein either or both of vWf and        fibrin(ogen) are immobilized to the solid support via an        antibody that specifically binds vWf or fVIII, respectively.    -   12. The method of paragraph 10 wherein the solid support        comprises a column matrix.    -   13. The method of paragraph 10 wherein the contacting with the        solid support comprises passing the sample over a column        comprising the solid support.    -   14. The method of paragraph 10 wherein the solid support        comprises Sepharose or polystyrene beads.    -   15. The method of paragraph 10 wherein after the contacting, the        solid support is washed with a buffer solution to remove unbound        fVIII.    -   16. The method of paragraph 15 wherein washing of the solid        support comprises a gradient sedimentation step.    -   17. A method of measuring the partitioning of fVIII between        binding to von Willebrand factor and binding to fibrin(ogen) in        a liquid sample, the method comprising contacting a solid        support comprising fibrinogen or fibrin with the sample,        removing the liquid sample from the solid support, and measuring        fVIII activity bound to the support versus fVIII activity        remaining in the liquid sample, wherein fVIII bound to the        support partitions with fibrin(ogen) and fVIII remaining in        solution is free or partitions with von Willebrand factor.    -   18. The method of paragraph 17 wherein either or both of vWf and        fibrin(ogen) are immobilized to the solid support via an        antibody that specifically binds vWf or fVIII, respectively.    -   19. The method of paragraph 17 wherein the solid support        comprises a column matrix.    -   20. The method of paragraph 17 wherein the contacting with the        solid support comprises passing the sample over a column        comprising the solid support.    -   21. The method of paragraph 17 wherein the solid support        comprises magnetic beads, agarose beads or polystyrene beads.    -   22. The method of paragraph 17 wherein after the contacting, the        solid support is washed with a buffer solution to remove unbound        fVIII.    -   23. The method of paragraph 22 wherein washing of the solid        support comprises a gradient sedimentation step.    -   24. A method of measuring the partitioning of fVIII between        binding to von Willebrand factor and binding to fibrin(ogen) in        a liquid sample, the method comprising contacting a solid        support comprising an antibody that specifically binds factor        VIII with the sample, removing the solid support, and measuring        the amount of von Willebrand factor and the amount of        fibrin(ogen) bound to the support together with factor VIII.    -   25. The method of paragraph 24 wherein the solid support        comprises magnetic beads.    -   26. The method of paragraph 24 wherein the solid support        comprises agarose beads.    -   27. The method of paragraph 24 wherein the solid support        comprises polystyrene beads.    -   28. The method of paragraph 24 wherein the von Willebrand factor        and fibrin(ogen) bound is measured by flow cytometry using        fluorescently labeled antibodies that specifically bind von        Willebrand factor and fibrin(ogen), respectively.    -   29. The method of paragraph 24 wherein the solid support is        separated from the liquid sample by magnetic force.    -   30. The method of paragraph 24 wherein the solid support is        separated from the liquid by sedimentation.    -   31. A kit for measuring fVIII activity, the kit comprising:        fibrin(ogen);    -   a platelet membrane-comprising composition;    -   Factor IXa;    -   Factor X; and    -   packaging materials therefor.    -   32. The kit of paragraph 31 further comprising a chromogenic fXa        substrate.    -   33. The kit of paragraph 31 wherein the fibrin(ogen) is in        association with the platelet membrane-comprising composition.    -   34. The kit of paragraph 31 wherein the platelet        membrane-comprising composition comprises activated platelets.    -   35. The kit of paragraph 31 wherein the platelet        membrane-comprising composition comprises activated gpIIbIIIa        incorporated into a membrane composition.    -   36. A kit for detecting the fraction of factor VIII bound to von        Willebrand factor vs. fibrin(ogen) wherein the kit comprises:    -   a solid support having monoclonal antibodies that specifically        bind fVIII immobilized thereupon;    -   a device for separating the beads from blood or plasma,    -   fluorescently labeled detection antibodies that specifically        bind fibrin(ogen) and von Willebrand factor, respectively;    -   calibrated control beads; and    -   packaging materials therefor.

EXAMPLES Example 1 Thrombin-Stimulated Platelets have Functional BindingSites for fVIII that are Distinct from Phosphatidylserine

FVIII functions as a co-factor for fIXa on the membranes of stimulatedplatelets. Binding sites for fVIII are expressed at two levels; thrombininduces 3,000-20,000 sites/platelet while the combination of collagenand thrombin or A28137 induce >50,000 sites/platelet.

The inventors hypothesized that binding sites for fVIII onthrombin-stimulated platelets, are distinct from phosphatidylserine(PS), while those on maximally stimulated platelets are predominantlyPS-containing sites. The hypothesis was based on the ideas that 1)epitopes on fVIII interact with the non-PS sites and 2) a macromoleculeor a macromolecule complex comprises the binding sites onthrombin-stimulated platelets.

Methods

Platelets were purified on a discontinuous density gradient and bindingof fluorescein-labeled fVIII (fVIII-fluor) to platelets and Superosebeads was measured by flow cytometry using a Becton DickinsonLSR-Fortessa flow cytometer. FVIII activity was measured in adiscontinuous factor Xase assay using extruded phospholipid vesicles ofcomposition PS:PE:PC 4:20:76 or platelets as the membrane source.Oligomeric fibrin was immobilized by incubating thrombin, 1 u/ml, withfibrinogen, 10 μg/ml for 10 min without mixing prior to addition of59D8-Superose beads. Binding of fVIII-4 Ala to platelets was measured incomplex with Alexa-488 labeled mAb GMA-8021, against the A2 domain.

Results

Lactadherin, a phosphatidyl-L-serine-binding protein, competed for 97%of fVIII-fluorescein (fVIII-fluor) binding sites on A23187-stimulatedplatelets but only 30% of binding sites on thrombin-stimulatedplatelets. Unlabeled fVIII competed with fVIII-fluor for all bindingsites. A fVIII C2 domain mutant, with no measurable phospholipidbinding—M2199A/F2200A/L2251A/L2252A (fVIII-4Ala) bound to only3,000-5,000 sites on platelets stimulated with A23187 but to a similarnumber on thrombin-stimulated platelets with a KD of 7 nM. These dataindicate that non-PS sites are dominant on thrombin-stimulated plateletsbut that PS-containing sites comprise at least 95% of sites onA23187-stimulated platelets.

The inventors evaluated a panel of monoclonal antibodies (mAbs) againstthe fVIII-C2 domain for platelet-specific inhibition of binding andfunction. mAbs ESH4 and 154, with overlapping epitopes, blocked bindingof fVIII to thrombin-stimulated platelets but only decreased affinityfor PS-containing membranes. In 1-stage and 2-stage commercial aPTTassays ESH4 inhibited 28-33% of fVIII activity. In contrast, ESH4inhibited 80% of fVIII activity on thrombin-stimulated platelets. mAb'sESH8 and G99, with partially overlapping epitopes decreased the affinityof fVIII-fluor for thrombin-stimulated platelets approx. 70% but had noeffect on phospholipid binding ESH8 inhibited 58±8% of fVIII activity onthrombin-stimulated platelets but had no effect on activity supported byphospholipid vesicles.

Because oligomeric fibrin is required for expression of most fVIIIbinding sites on thrombin-stimulated platelets (Phillips et al 2004; JTH2:1806) the inventors hypothesized that oligomeric, platelet-boundfibrin is a constituent of fVIII binding sites. fVIII-fluor bound tofibrin monomers and oligomers immobilized on mAb 59D8-Superose, detectedin solution by flow cytometry. Both ESH4 and ESH8 monoclonal antibodiesselectively interfere with fVIII binding to fibrin; it is noted thatwhile ESH8 interferes with fVIII binding to fibrin, this antibody doesnot interfere with fVIII binding to vWF.

These data indicate that thrombin-stimulated platelets bind fVIII via anon-PS binding site and that the binding is mediated by epitopes thathave greater functional importance on platelets than on phospholipidvesicles. Platelet-bound oligomeric fibrin is a candidate for the non-PSbinding site. These findings have clinical relevance to detection ofinhibitory antibodies against fVIII.

TABLE 1 Effect of fibrin on parameters of factor Xase complex V_(Max)K_(D) Apparent K_(D) Apparent K_(M) [fXa formed] fVIIIa <-> fIXa[Phospholipid*] [fX] nM nM/5 min. nM μM Control + 57 ± 10 2.5 ± 0.1 19 ±2 37 ± 4 fibrin 32 ± 5  3.7 ± 0.2  5 ± 1 32 ± 6 Parameters wereevaluated in the presence or absence of 20 μg/ml fibrin. Fibrinogen wasexposed to 0.1 unit/ml thrombin for 2 min prior to addition of hirudin.Concentrations of each component were varied to evaluate the K_(M),V_(Max), and apparent dissociation constants. *Extruded phospholipidvesicles had composition of PS:PE:PC 4:20:76.

The inventors' data indicate that platelets have two classes of fVIIIbinding sites. Platelet stimulation by calcium ionophore, anon-physiologic agonist, leads to exposure of many binding sitescomprised primarily of exposed phosphatidylserine. Platelets stimulatedby thrombin, a physiologic agonist, have binding sites that are notcomprised primarily of phosphatidylserine. FVIII activity supported bythrombin-stimulated platelets is inhibited to a different degree by someinhibitory antibodies than fVIII activity supported by plateletsstimulated by calcium ionophore.

Lactadherin, a phosphatidyl-L-serine binding protein blocks binding offVIII to greater than 95% of binding sites on calcium ionophorestimulated platelets but only 30% of sites on platelets stimulated bythrombin

An engineered fVIII molecule (fVIII-4 Ala) has defective phospholipidbinding. Yet this molecule retains binding to thrombin-stimulatedplatelets confirming that the binding sites are not comprised ofphospholipid. It has 95% reduction in binding to platelets stimulatedwith calcium ionophore.

These data are consistent with a model where platelet-bound fibrinserves as the primary binding site for fVIII on thrombin-stimulatedplatelets.

Summary

The inventors' data indicate that fVIII binds to: (i) fibrinogenimmobilized on anti-fibrinogen antibodies supported by Superose beads.The KD is approximately 2 nM; (ii) fVIII-4 Ala binds to immobilizedfibrinogen with lower affinity, and (iii) fVIII binds to suspendedfibrinogen, not influenced by binding to antibodies or to SUPEROSE™agarose beads.

The data further indicate that fVIII binds to: Oligomeric fibrin boundto an anti-fibrin antibody supported by Superose beads.

In addition, the data further indicate that activity of pure fVIII isincreased by fibrinogen when (i) fVIII is activated by factor Xa,without conversion of fibrinogen to fibrin; the degree of activation is2-6 fold, and (ii) when fVIII is activated by thrombin and fibrinogen isconverted to fibrin. Pure fVIII is not increased by fibrinogen whenfVIII is cleaved and released from the fVIII:von Willebrand complex bythrombin.

The data further indicate that: (i) von Willebrand factor competes withfibrinogen for fVIII binding, and (ii) von Willebrand factor decreasesthe activity of fVIII in the presence of factor Xa with the degree ofinhibition corresponding to the predicted binding of fVIII to vonWillebrand factor.

This discovery establishes a distinct pathway for fVIII activation andfunction that does not rely on prior generation of thrombin and does notrequire the presence of membranes of high phosphatidylserine content.

Applications

Provided herein are methods and assays for measuring the biologicalactivity of fVIII using platelets, or a platelet-like substance, toreplace phospholipids as a membrane source. An assay of this natureprovides a better indication of the biologically relevant activity offVIII as compared to the standard fVIII assay that activates plateletsusing a non-physiological agonist (e.g., a calcium ionophore). Alsoprovided herein are screening assays for agents that modulate fVIIIactivity (e.g., recombinant fVIII).

Example 2 Platelet Binding Sites for Factor VIII in Relation to Fibrinand Phosphatidylserine

It is demonstrated herein that lactadherin competes for 20-25% of factorVIII binding sites on thrombin-stimulated platelets while unlabelledfactor VIII competes for >70% of these sites. Thus, most factor VIIIbinding sites do not rely on exposed phosphatidylserine. LW-fibrinattached to Superose beads resembled platelets in having high affinityfactor VIII binding that was prevented by von Willebrand factor.LMW-fibrin enhanced activity of factor VIII by 2-3 fold in a factor Xaseassay. Anti-factor VIII C2 domain mAb's ESH4 and ESH8 inhibited thebinding of factor VIII to fibrin, activity enhancement by fibrin,binding of factor VIII to platelets, and >90% of factor VIII activity inan activated platelet-based clotting assay. These results indicate thatplatelet-bound fibrin functions as a component of factor VIII bindingsites and that activity on these sites is inhibited by antibodies in aqualitatively distinct manner.

Factor VIII binds to platelet membranes where it serves as a cofactorfor the enzyme, factor IXa in the intrinsic factor Xase complex (1, 2),which converts the zymogen factor X to factor Xa (3, 4). The importanceof the factor Xase complex is illustrated by the disease haemophilia, inwhich deficiency of factor VIII (haemophilia A) or factor IX(haemophilia B) leads to life-threatening bleeding. In spite of thecentral importance of factor VIII, the platelet membrane binding siteshave been only partially characterized.

Factor VIII circulates in plasma in a non-covalent complex with vonWillebrand factor (VWF). Binding is mediated by the same motifs thatbind platelet and phospholipid membranes (5, 6). After dissociation fromVWF, factor binds specifically to membranes containingphosphatidylserine (PS), which is exposed on the platelet membrane inresponse to stimulation by several agonists (1, 6). The residualuncertainty about the identity of platelet binding sites relates to thequantity of PS exposed following stimulation by physiologic agonists andthe availability of specific reagents to block the exposed PS. Thrombinstimulates platelets to expose limited PS, resulting in an outermembrane composition of 1-4% PS (7, 8). This amount of PS may remainbelow the threshold to support the observed expression of 200-1600binding sites/platelet (9, 10). The combination of thrombin andcollagen, or higher concentrations of the calcium ionophore, A23187,lead to complete PS exposure with membrane composition estimated at12-15% PS (7, 8). Under these conditions, PS is thought to be a criticalcomponent of most of the >10,000 factor VIII binding sites exposed perplatelet (11, 12).

Factor VIII has a domain structure of A1-a1-A2-a2-B-a3-A3-C1-C2, wherea1, a2 and a3 are spacer regions. (13) The C1 and C2 domains mediatemembrane binding (14-18). The factor VIII C domains share similarsequence and structure with the C domains of factor V (19-21) andlactadherin (22) a milk fat globule membrane protein. Like factor VIII,both factor V and lactadherin preferentially bind tophosphatidylserine-containing membranes (23, 24). The membrane-bindingrole of the protruding hydrophobic amino acids of the factor VIII (17),factor V (25, 26), and lactadherin (27) C2 domains has been confirmed bysite-directed mutagenesis. A factor VIII mutant, factor VIII-4Ala, withhydrophobic spike amino acids changed (M2199A/F2200A, L2251A/L2252A) hasless than 1% residual binding to and activity on synthetic,phosphatidylserine-containing membranes (17). This factor VIII mutanthas been used in the present study to test the hypothesis the plateletshave binding sites not determined by membrane phospholipid.

Binding of soluble fibrin to the α_(IIb)β₃ integrin onthrombin-stimulated platelets increases the number of factor VIIIbinding sites by 3-8 fold (28). However, factor VIII did not bind tofibrin adsorbed to polystyrene beads, indicating that factor VIII (i)does not bind directly to fibrin, (ii) the fibrin binding site wasaltered by contact with polystyrene, or (iii) that fibrin must be boundto the α_(IIb)β₃ integrin. Described herein is the evaluation of thepossibility that factor VIII(a) may bind directly to fibrin and thatplatelet-bound fibrin may be a component of the platelet binding sitesfor factor VIII.

Haemophilia A is treated by infusing purified plasma factor VIII intodeficient patients. However, such treatment can result in the productionof antibodies against factor VIII that impair function (29). Nearly halfof inhibitory antibodies are directed against the C2 domain and manyinterfere with binding to phospholipid membranes and VWF. Monoclonalantibodies directed against the C2 domain mimic clinical inhibitoryantibodies (30). ESH4 and ESH8 are two prototype anti-C2 antibodies withnon-overlapping epitopes (31). ESH4 partially blocks binding tophosphatidylserine-containing membranes and binding to von Willebrandfactor (30, 32). ESH8 doesn't inhibit factor VIII activity supported byPLV (32, 33), but slows the dissociation of activated factor VIII fromvon Willebrand factor (33). Described herein is the evaluation of theeffect of ESH4 and ESH8 on factor VIII binding to fibrin and plateletsand the extent to which the inhibitory effect on platelet-based factorVIII activity differs from phospholipid vesicle-based activity.

Materials

Materials: Porcine brain phosphatidylserine (PS), eggphosphatidylethanolamine (PE), and egg phosphatidylcholine (PC) werepurchased from Avanti Polar Lipids. Calcium ionophore A23187, OptiPrep,thrombin receptor agonist peptides (TRAP) for PAR1 (SFLLRNPNDKYQPF) andPAR4 (AYPGKF-amide) were purchased from Sigma Aldrich. Purified humanfibrinogen (unless otherwise specified), factor X, factor Xa, factorIXa, and thrombin were purchased from Enzyme Research Labs. FactorVIII-free VWF, corn trypsin inhibitor, and human factor XIa werepurchased from Haematologic Technologies. Chromogenic substrate (S-2765)was purchased from Diapharma. Monoclonal antibodies ESH4 and ESH8 werepurchased from American Diagnostica. Antibody GMA-8021 was purchasedfrom Green Mountain Antibodies. Polyclonal antibodies to fibrinogen(ab6666), fibronectin (ab299), plasminogen (ab6189), and VWF (ab96340)were from Abcam. Anti-fibrin fragment E polyclonal antibody (F4197-40)was purchased from US Biological. Another anti-fibrinogen antibody wasfrom Dako (A0080).

Methods

Proteins: Lipid-free bovine lactadherin was purified as described (24).The factor VIII C2 domain (fVIII-C2) was produced, purified, and storedas described. (18) Factor VIII was labeled with fluorescein maleimide asdescribed (11). The ratio of fluorescein:factor VIII ranged from 0.6/1to 1.2/1 for studies in this report. GMA8021 was labeled withfluorescein isothiocyanate using a standard protocol.

Flow cytometry measurement of factor VIII binding to platelets.Platelets from human volunteers were purified as described (28) anddiluted in Tyrode's buffer (138 mM NaCl, 2.7 mM KCl, 3.3 mM NaH₂PO₄, 1mM MgCl₂, 1% dextrose, 0.2% bovine serum albumin, 15 mM HEPES, pH 7.4)Binding of fluorescein-labeled factor VIII was measured by flowcytometry as previously described using a Becton Dickinson LSR-Fortessa™flow cytometer.

Factor VIII binding to fibrin: Polyclonal anti-fibrinogen and mAb 59D8were covalently coupled to batches of cyanogen bromide-activatedSuperose™ beads with an antibody/Superose™ ratio of 1.25 mg/mL asdescribed (34). Fibrinogen, 10 μg/ml, was incubated with the Superose™beads for 20 min. at 22° C. with 1 u/mL thrombin. Hirudin (3 u/mL) wasadded and excess fibrin was removed by sedimenting the Superose beads500 g×30 s. Fibrin-Superose™ beads were resuspended in 50 mM Tris pH7.85, 150 mM NaCl, 0.01% Tween 80, 0.1% BSA in a 2:1 ratio with Optiprepand incubated with factor VIII-fluor and competitors or inhibitors asindicated. In some experiments factor VIII-fluor was mixed with mAb'sESH4 or ESH8, VWF, or factor VIII-C2 prior to mixing withSuperose™-fibrin beads, as indicated. Bound factor VIII-fluor wasevaluated by flow cytometry on an LSR-Fortessa™ flow cytometer. Dataacquisition was triggered by forward light scatter values characteristicof Superose™ beads. Data was evaluated as the geometric meanfluorescence of the dominant population (typically >90% of events).

Factor VIII activity: Factor VIII activity in the factor Xase complexwas measured with two-step amidolytic substrate assays as described(15). Phospholipid vesicles were prepared by high-pressure extrusionthrough two-stacked polycarbonate membranes with laser-etched pores asdescribed (35). Added fibrinogen was activated with thrombinconcurrently with the factor VIII. One and two-stage factor VIIIactivity was measured in an aPTT assay (Helena Labs, aPTT-SA reagent)and in a 2-stage chromogenic assay (DiaPharma). One stage assays wereperformed, according to package instructions, with the BBL fibrometer.The chromogenic assay was performed according to manufacturerinstructions with chromogenic substrate development read on a VersaMax™microplate reader in kinetic mode.

Activated platelet time clotting assay: Purified platelets (28) weremixed with factor VIII deficient plasma supplemented with 25 μg/mL corntrypsin inhibitor to give a composition of 1×10⁸ platelets/ml. Variousconcentrations of factor VIII were mixed with 10% normal plasma inTyrode's buffer lacking phosphate and incubated for 1 hr at 22° C. priorto evaluating factor VIII activity. For the activated platelet timeassay, 100 μl of reconstituted platelet-rich-plasma was mixed with 100μl of factor VIII±inhibitory antibody for 1 min at 37° C. The clottingreaction was started by adding 100 μl of a mixture containing fXIa (200pM), TRAP-PAR1 (30 μM), TRAP-PAR4 (300 μM), and CaCl (15 mM) inphosphate-free Tyrode's albumin buffer. Fibrin strand formation wasmeasured with a BBL fibrometers in 300 μl sample cups. Experiments wereperformed in triplicate.

Results

To test the hypothesis that platelets have non-phospholipid bindingsites or receptors for factor VIII (9, 36), competition by lactadherinfor factor VIII binding sites was evaluated (FIGS. 1A-1B). FactorVIII-fluor bound to many binding sites on platelets stimulated by A23187and thrombin (FIG. 1A) consistent with the large number of binding sitespredicted from extensive PS exposure. Addition of lactadherin, at aconcentration that blocks >99% of factor VIII binding to PS-containingsynthetic membranes (35), blocked approx. 95% of factor VIII binding tostimulated platelets. However, factor VIII-fluor still bound saturablyto approx. 5% of binding sites, corresponding to more than 1,000sites/platelet. This indicates that PS is not a critical component of3-5% of the sites.

Thrombin, without A23187, stimulated expression of 450-1800 bindingsites, as previously reported (28). Lactadherin competed for 20-25% ofthese binding sites (FIG. 1B) indicating that PS is a critical componentof some binding sites, consistent with the previously reported limitedPS exposure (7, 8, 37, 38). In contrast to lactadherin, unlabeled factorVIII competed for >70% of factor VIII binding sites, indicating specificsites for factor VIII that don't rely on PS.

Binding of a factor VIII mutant with defective phospholipid binding wasevaluated. Factor VIII-4Ala (M2199A/F2200A, L2251A/L2252A) has <1%residual phospholipid membrane affinity (17) (FIGS. 2A-2B). Binding offactor VIII-4Ala was reduced approx. 99% on platelets stimulated withA23187 and thrombin (FIG. 2A). In contrast, factor VIII-4Ala bound toapprox. 50% of binding sites on platelets stimulated by thrombin (FIG.2B). These results confirm that the phospholipid binding motif of thefactor VIII C2 domain is not critical for at least 50% of binding siteson thrombin-stimulated platelets.

Binding of soluble fibrin to the α_(IIb)β₃ integrin results in a 3-6fold increase in factor VIII binding sites on thrombin-stimulatedplatelets (28). It was asked whether soluble fibrin bound to a plateletmight serve as a binding site (FIGS. 10A-10C). Accordingly, solublefibrin (39) was prepared in the presence of anti-fibrinogen antibodiescoupled to Superose™. Immobilization of fibrin was verified with afluorescein-labeled an anti-fibrinogen/anti-fibrin antibody. FactorVIII-fluor bound to fibrin-Superose with half-maximal binding at 1-2 nM(FIG. 4A). Factor VIII-fluor bound to fibrin with similar affinity whenfibrin was immobilized on Superose-59D8, an antibody n-terminus of thefibrin β chain (40) confirming that fibrin binds factor VIII. FactorVIII-4Ala also bound to immobilized fibrin (data not shown). Thequantity bound was approx. 50% of wild type factor VIII at 2 nM. Thesedata indicate that factor VIII binds to fibrin and that thefibrin-interactive residues differ from those that are critical forbinding to PS-containing membranes.

VWF prevented binding of factor VIII to immobilized fibrin, similar toinhibition of factor VIII binding to thrombin-stimulated platelets (FIG.4B) (5). Because factor VIII binding to platelets and to VWF ismediated, in part, by the C2 domain it was asked whether the isolated C2domain (fVIII-C2) competes with factor VIII for binding to fibrin (FIG.4C). FVIII-C2 competed with factor VIII for binding to fibrin with 50%competition at approx. 0.1 μM factor VIII-C2. The competition studieswith factor VIII-C2 were performed in a low salt buffer, similar tobuffer conditions required for fVIII-C2 to bind phospholipid membranes(18). These results indicate that factor VIII binding to fibrin issimilar to binding to platelets with regard to affinity, prevention byVWF and participation of the C2 domain.

Because factor VIII binds to soluble fibrin it asked what effect fibrinhas on the activity of factor VIII (FIGS. 5A-5B). Soluble fibrinincreased activity of the factor Xase complex approx. 2.7-fold with ahalf-maximal increase at 5-10 μg/ml fibrin (FIG. 5A). At fibrinconcentrations exceeding 200 μg/ml factor Xase activity decreased (notshown) reaching baseline at approx. 500 μg/ml. The size of the fibrinenhancement was inversely related to the PS content of PLV's supportingthe reaction (FIG. 5B). The degree of enhancement was 2.7-fold on PLVwith 4% PS and 1.8-fold or less on PLV with 8% and 16% PS. These resultsindicate that soluble fibrin increases factor Xase activity on PLV withPS content similar to thrombin-stimulated platelets.

The constituents of the factor Xase complex were varied systematicallyto determine which steady state kinetic parameters of the factor Xasecomplex were altered (Table I). The results indicated that solublefibrin increases the apparent affinity of factor Villa for factor IXa byabout 4-fold. In addition, the V_(max) increased by 50% and the K_(M)decreases by about 50%. Thus, the largest effect on parameters of steadystate kinetics is on the apparent affinity of factor Villa for factorIXa.

The data presented herein indicates that soluble fibrin has relativelyfew binding sites that increase factor VIII activity. Maximalenhancement of factor VIII activity occurs with a fibrin monomer/factorVIII ratio of approx. 10-30 (FIG. 5A). Modifying the experimentalprotocol to increase or decrease the size of soluble fibrin units by 2-6fold did not affect the effective concentration (data not shown). Thus,it appears that the ratio of fibrin monomer/factor VIII activityenhancing site is determined by a unique property of a small fraction offibrinogen molecules.

Because the C2 domain is implicated in binding to fibrin by the datapresented herein, it was asked whether mAb's against the C2 domaininhibit this interaction (FIG. 6A). Both ESH8 and ESH4 inhibit factorVIII binding to fibrin (FIG. 6A). It was then tested whether theantibodies blocked the fibrin-mediated increase in factor Xase activity(FIG. 6B). ESH4 decreased, but did not entirely prevent, activitysupported by PLV, as previously observed (15). However, the residualactivity in the presence of ESH4 was not enhanced by addition of solublefibrin (FIG. 6B). As previously reported, ESH8 did not inhibit factorVIII activity in the absence of fibrin (33) but did prevent the increasemediated by fibrin (FIG. 6B).

ESH8 decreased the number of binding sites recognized by factor VIII onthrombin stimulated platelets by approx. 70% (FIG. 7A) similar to thenon-phospholipid fraction of binding sites (FIG. 1B). ESH4 alsoinhibited factor VIII binding to thrombin-stimulated platelets (FIG.7B). The degree of inhibition was greater, consistent with the capacityof ESH4 to decrease affinity for PS-containing membranes (33) as well asfibrin. ESH8 inhibited 80% of platelet-dependent factor Xase activity(FIG. 7C). In a similar manner, ESH4 decreased activity by 70-85%. Thus,the inhibition of factor Xase activity parallels the inhibition offactor VIII binding to thrombin-stimulated platelets. However, theinhibition of platelet-based activity by ESH8 is in marked contrast withits lack of inhibition of factor VIII activity supported byPS-containing membranes (33).

To evaluate the importance of the ESH4 and ESH8 epitopes in a morephysiologic system an activated platelet clotting time was developed(FIGS. 10A-10C). Purified platelets were re-constituted with factorVIII-deficient plasma supplemented with various concentrations of factorVIII. The reaction was initiated by simultaneous addition of factor XIa,thrombin receptor activation peptides for PAR1 and PAR4, and Ca⁺⁺. Theresults indicated a log-linear relationship between factor VIIIconcentration and time to fibrin strand formation over a wide factorVIII concentration range (FIG. 10A). When ESH8 was incubated with 1 u/mlfactor VIII the delay in fibrin strand formation was consistent with a97% reduction in factor VIII activity (FIGS. 10A, 10C). Because ESH8inhibits release of factor Villa from VWF (33), experiments with ESH8were also performed with VWF-deficient plasma. ESH8 inhibited 93% offactor VIII activity in the absence of plasma VWF. This contrasts withthe degree of inhibition by ESH8 in standard aPTT and 2-stage factorVIII activity assays (FIG. 10C) where there is less inhibition in thepresence of VWF and no inhibition in the absence of VWF.

ESH4 inhibited 99% of factor VIII activity in the activated platelettime assay (FIG. 10A). In contrast, the degree of inhibition ranged from30% to 70% in commercial aPTT and 2-stage assays (FIG. 10B). When ESH4was incubated with factor VIII in VWF-deficient plasma the degree ofinhibition was 99.7% (FIG. 10B-inset). Without wishing to be bound bytheory, ESH4 and VWF may compete for overlapping epitopes on factor VIIIand VWF likely protected a fraction of factor VIII from interacting withESH4. Overall, the results indicate that the degree of inhibition in aplatelet and plasma-based system is better predicted by factor VIIIbinding to platelets and to fibrin than to phospholipid vesicles.

Discussion

The data presented herein demonstrate that stimulated platelets havefactor VIII(a) binding sites that are distinct from membrane PS. FactorVIII binds to soluble fibrin and the binding characteristics of thatinteraction parallel the interaction with the non-PS binding sites ofplatelets. Notably, factor VIII has similar affinity for fibrin andbinding is blocked by VWF in a similar manner. Further, inhibition offactor VIII binding to fibrin predicts the inhibitory activity of twomonoclonal antibodies to factor VIII in platelet-based assays. Thus,they support a hypothesis that platelet-bound fibrin is a component ofnon-PS platelet binding sites.

It is demonstrated herein that lactadherin, a PS-binding protein thatcompetes for >99% of sites recognized by factor VIII (35), does notcompete for most sites on thrombin-stimulated platelets. Further, it isdemonstrated herein that a factor VIII mutant with severely impairedphospholipid affinity (17) retains binding to thrombin-stimulatedplatelets.

Without wishing to be bound by theory, it is contemplated herein that PSis not necessarily required for full activity of factor VIII. Althoughlactadherin did not compete for a class of factor VIII binding sites, itdid diminish activity by >99%, as previously reported (38).

Soluble fibrin binding to the α_(IIb)β₃ integrin of platelets increasesbinding sites for factor VIIIa (28). Fibrin adsorbed to polystyrenebeads had no detectible binding to factor VIII. As described herein,soluble fibrin bound to an antibody on a porous Superose matrix doesbind factor VIII. Without wishing to be bound by theory, it iscontemplated herein that soluble fibrin bound to the α_(IIb)β₃ integrinmay support factor VIII binding. The binding to these sites wasdiminished when beads were sedimented in multiple washes. This indicatesthat the binding surfaces can adhere to other fibrin molecules or aresubject to conformational change.

In the absence of VWF, ESH8 doesn't inhibit activity of factor VIII onPS-containing membranes (33). Thus, inhibition of factor VIII binding tofibrin (FIGS. 6A-6B), inhibition of factor VIII binding to platelets(FIG. 7A) and inhibition of platelet-based procoagulant activity (FIGS.10A-10C) appear to identify a distinct function of the ESH8 epitope thathas not been reported.

The importance of the ESH4 epitope for membrane binding is modest whenthe PS content of vesicles is above 15% (15). Thus, it is not surprisingthat ESH4 causes only modest inhibition of factor VIII activity in thecommercial 1 and 2-stage factor VIII activity assays that havesaturating concentrations of phospholipid vesicles with high PS content(FIG. 10B). In contrast, ESH4 blocked binding to fibrin and >80% ofbinding sites on thrombin-stimulated platelets. This correlated to 85%reduction of platelet factor Xase activity (FIG. 10C) and 98-99%reduction of platelet procoagulant activity (FIG. 10B). In VWF-deficientplasma the inhibition of platelet procoagulant activity was greater.This indicates that the non-phospholipid factor VIII binding sites havegreater importance in plasma coagulation than the 20-25% plateletbinding sites that are mediated by phospholipid (FIG. 1B).

The activated platelet time assay utilized herein differs from a priorplatelet-based coagulation assay in that platelets were activated at theoutset of the reaction by peptides that stimulate platelets via PAR1 andPAR4 (42). The results with the present assay contrast with results fromreported factor VIII assays. First, the range over which fibrin strandformation had a log-linear relationship of factor VIII concentration tocoagulation time of 0.0003-0.3 units/ml. In contrast, established factorVIII assays have a factor VIII-time log-linear range of 0.01-0.3units/ml (43). Thus, a coagulation assay based on the activated plateletmembrane rather than phospholipid vesicles may have potential as thebasis of a clinical assay with a broader range. In addition, thesensitivity of the assay to factor VIII inhibition by ESH4 and ESH8differed by >10-fold compared to commercially available 1-stage and2-stage assays. These data indicate that an assay in which factor VIIIactivity is supported by the activated platelet membrane can provideclinical information for patients who have developed inhibitorantibodies.

In summary, the data presented herein demonstrate that platelets havenon-PS binding sites for factor VIII. Soluble fibrin is required toconstitute these sites and fibrin binds factor VIII with properties thatindicates that fibrin or a complex of fibrin with another molecule isthe best candidate for the non-phospholipid site. Activity of factorVIII on the non-PS platelet sites has qualitatively differentsusceptibility to anti-factor VIII antibodies in a platelet-basedcoagulation assay.

Supplemental Data

Experiments with factor VIII-4Ala differed in that factor VIII-4Ala waslabeled by incubation with mAb GMA8021-fluor for 1 hr at a ratio of 4:1.For these experiments wild type factor VIII was labeled in the samemanner as a control. Recombinant factor VIII-4Ala was prepared bymutagenesis within the mammalian expression vector pMT2 as described(45). Conditioned medium from transfected COS-1 cells was harvested at64 h post-transfection in the presence of 10% fetal bovine serum andpurified by immunoaffinity chromatography as described (17). Eachpreparation yielded 1.5-3.0 μg of protein. The purified protein wasstored at −70° C. until use. The factor VIII C2 domain was produced inE. coli, purified, and stored as described (18).

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What is claimed herein:
 1. A kit for measuring fVIII activity, the kitcomprising: fibrin or fibrinogen; a platelet membrane-comprisingcomposition; Factor IXa; Factor X; a solid support having monoclonalantibodies that specifically bind fVIII immobilized thereupon; andpackaging materials therefor.
 2. The kit of claim 1 further comprising achromogenic fXa substrate.
 3. The kit of claim 1 wherein said fibrin orfibrinogen is in association with said platelet membrane-comprisingcomposition.
 4. The kit of claim 1, wherein said plateletmembrane-comprising composition comprises activated gpIIbIIIaincorporated into a membrane composition.
 5. The kit of claim 1, furthercomprising instructions for a method of measuring fVIII activity,wherein the method comprises the steps of: contacting a sample in whichfVIII is to be measured with (i) fibrin or fibrinogen and (ii) aplatelet membrane-comprising composition, under conditions that permitbinding of fibrin or fibrinogen to said platelet membrane-comprisingcomposition and permit binding of fVIII in said sample to said fibrin orfibrinogen, and detecting activity of said fVIII contacting is performedin the presence of Factor IXa (fIXa) and Factor X (fX).
 6. The kit ofclaim 5, wherein the instructions further describe methods for detectingactivity of fVIII comprising detecting cleavage of a chromogenic fXasubstrate by fXa.
 7. The kit of claim 5, wherein the instructionsfurther describe a method of detecting activity of fVIII comprising aplasma-based clotting assay and/or use of a fibrometer and/or use of athromboelastometry device.